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Evidence for Circulation of the Rift Valley Fever Virusamong Livestock in the Union of Comoros

Matthieu Roger, Marina Beral, Séverine Licciardi, Miradje Soule,Abdourahime Faharoudine, Coralie Foray, Marie-Marie Olive, Marianne

Maquart, Abdouroihamane Soulaimane, Ahmed Madi Kassim, et al.

To cite this version:Matthieu Roger, Marina Beral, Séverine Licciardi, Miradje Soule, Abdourahime Faharoudine, et al..Evidence for Circulation of the Rift Valley Fever Virus among Livestock in the Union of Comoros.PLoS Neglected Tropical Diseases, Public Library of Science, 2014, 8 (7), pp.e3045. �10.1371/jour-nal.pntd.0003045�. �hal-01274567�

https://hal.univ-reunion.fr/hal-01274567

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https://hal.archives-ouvertes.fr

Evidence for Circulation of the Rift Valley Fever Virusamong Livestock in the Union of ComorosMatthieu Roger1,2,3.*, Marina Beral1,2,3., Severine Licciardi3., Miradje Soule4,

Abdourahime Faharoudine4, Coralie Foray1,2,3, Marie-Marie Olive1,2,3,5, Marianne Maquart1,2,3,

Abdouroihamane Soulaimane4, Ahmed Madi Kassim4, Catherine Cetre-Sossah1,2,3, Eric Cardinale1,2,3

1 Centre de Cooperation Internationale en Recherche Agronomique pour le Developpement (CIRAD), UMR 15 CMAEE, Sainte Clotilde, La Reunion, France, 2 Institut

National de la Recherche Agronomique (INRA), UMR 1309 CMAEE, Sainte Clotilde, La Reunion, France, 3 Centre de Recherche et de Veille sur les Maladies Emergentes dans

l’Ocean Indien (CRVOI), Plateforme de Recherche CYROI, Sainte Clotilde, La Reunion, France, 4 Vice-Presidence en Charge de l’Agriculture, l’Elevage, la Peche, l’Industrie,

l’Energie et l’Artisanat, Mde, Moroni, Union des Comores, 5 Unite de Virologie, Institut Pasteur de Madagascar, Antananarivo, Madagascar

Abstract

Rift Valley fever virus (RVFV) is an arthropod-borne phlebovirus reported to be circulating in most parts of Africa. Since 2009,RVFV has been suspected of continuously circulating in the Union of Comoros. To estimate the incidence of RVFV antibodyacquisition in the Comorian ruminant population, 191 young goats and cattle were selected in six distinct zones andsampled periodically from April 2010 to August 2011. We found an estimated incidence of RVFV antibody acquisition of17.5% (95% confidence interval (CI): [8.9–26.1]) with a significant difference between islands (8.2% in Grande Comore, 72.3%in Moheli and 5.8% in Anjouan). Simultaneously, a longitudinal entomological survey was conducted and ruminant trade-related information was collected. No RVFV RNA was detected out of the 1,568 blood-sucking caught insects, includingthree potential vectors of RVFV mosquito species. Our trade survey suggests that there is a continuous flow of live animalsfrom eastern Africa to the Union of Comoros and movements of ruminants between the three Comoro islands. Finally, across-sectional study was performed in August 2011 at the end of the follow-up. We found an estimated RVFV antibodyprevalence of 19.3% (95% CI: [15.6%–23.0%]). Our findings suggest a complex RVFV epidemiological cycle in the Union ofComoros with probable inter-islands differences in RVFV circulation patterns. Moheli, and potentially Anjouan, appear to beacting as endemic reservoir of infection whereas RVFV persistence in Grande Comore could be correlated with trade in liveanimals with the eastern coast of Africa. More data are needed to estimate the real impact of the disease on human healthand on the national economy.

Citation: Roger M, Beral M, Licciardi S, Soule M, Faharoudine A, et al. (2014) Evidence for Circulation of the Rift Valley Fever Virus among Livestock in the Union ofComoros. PLoS Negl Trop Dis 8(7): e3045. doi:10.1371/journal.pntd.0003045

Editor: Brian Bird, Centers for Disease Control and Prevention, United States of America

Received October 8, 2013; Accepted June 11, 2014; Published July 31, 2014

Copyright: � 2014 Roger et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This study was conducted in the framework of AnimalRisk-OI, a research program on emerging animal diseases in the Indian Ocean, funded by FEDERPOCT (European Union, Regional Council of Reunion and the French government). The study was partially funded by EU grant FP7-261504 EDENext and iscatalogued by the EDENext Steering Committee as EDENext000 (http://www.edenext.eu).The funders had no role in study design, data collection and analysis,decision to publish, or preparation of the manuscript.

Competing Interests: The authors have declared that no competing interests exist.

* Email: [email protected]

. These authors contributed equally to this work.

Introduction

Rift Valley fever (RVF) is an arthropod-borne zoonotic disease

caused by a RVF virus (RVFV), a member of the Phlebovirusgenus of the family Bunyaviridae [1]. RVFV causes significant

morbidity and mortality among sheep, goats, cattle and also affects

humans. In livestock, abortion storms and high mortality observed

among the younger animals cause significant economic losses

[2,3]. Humans are usually infected by contact with infectious

animal tissues through inhalation or aerosols generated by

slaughtering and necropsy [4]. Arthropod vectors play an

important role during the onset of epidemic and inter-epidemic

periods [5]. In endemic areas, RVFV is maintained in the

environment through an enzootic vertebrate-arthropod cycle [6].

RVFV has been isolated from many vectors in the field [7], such as

ticks and sand flies which are able to transmit the virus in

experimental conditions [8,9]. However, mosquitoes are the main

insects involved in the spread of RVFV during epidemics. RVFV

has been isolated from at least 40 species of mosquitoes belonging

to 8 genera but only some of them are susceptible and able to

transmit RVFV under laboratory conditions [10]. RVF is widely

present in Africa and has been spreading to Madagascar and the

Arabian Peninsula [11,12]. In 2007, RVF outbreaks were reported

in several eastern and southern African countries [13]. A few

weeks later, and for the first time, RVFV was detected in the

Comoros archipelago following the hospitalization of a young

Grande Comorian boy showing symptoms of severe encephalitis

[14]. In addition, during the 2008 and 2009 rainy seasons,

outbreaks due to RVFV strains imported from mainland Africa

were reported in Madagascar causing 59 confirmed human cases

and seven deaths [11,15].

In Mayotte, the French overseas territory that belongs to the

Comoros archipelago, a retrospective study conducted in 2008

confirmed the presence of the disease with 10 human cases

PLOS Neglected Tropical Diseases | www.plosntds.org 1 July 2014 | Volume 8 | Issue 7 | e3045


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infected with RVFV strains genetically closely linked to the 2006–

2007 Kenyan isolates [16]. It was also found that the Mayotte

livestock has been infected by RVFV prior to 2004 [17].

Regarding the Union of Comoros, in 2011, Roger et al. reported

widespread exposure of Comorian livestock with 32.8% of animals

shown to be RVFV-seropositive without any notifications of

massive abortions or abnormal mortality in the younger animals

by the Comorian Animal Health Services. However, the origin of

this infection remains unknown [18].

The Union of Comoros is located in the South West Indian

Ocean at the northern end of the Mozambique Channel and is

considered to be a gateway to islands in the Indian Ocean for

various infectious agents imported from mainland Africa. Since

2002, live ruminants are imported from Tanzania and have

entered the country without a period of quarantine or a clinical

examination [18]. Finally, in the past, animal trade has already

affected the country health status on several occasions, with regard

to many diseases, like blackleg in 1970 and 1995, the contagious

ecthyma in 1999, and the East Coast fever in 2003 and 2004 [19–

21].

Some of the Culicidae species described in the Comoros

archipelago [22] have already been shown to be involved in

RVFV transmission. The establishment of RVFV in the Union of

Comoros remains unconfirmed and the threat to the Comorian

population and neighboring countries needs to be considered.

The trade and resulting movements of ruminants, the compo-

sition and abundance of the vector population and many other

environmental and anthropological factors determine the nature

of the RVF viral cycle. In order to elucidate how RVFV persists in

the Union of Comoros, longitudinal and cross-sectional livestock

surveys were conducted between April 2010 and August 2011 in

six separate geographical zones. Mosquito populations were

categorized in parallel over the same period via a longitudinal

entomological survey. Additionally trade frequencies were ana-

lyzed, providing an estimate of regional ruminant flux and

allowing for evaluation of the risk associated with animal

importation, and the likelihood of RVFV persistence in the

Comoros islands.

Materials and Methods

Ethics statementThe research protocol was implemented with the approval of

the Vice-Presidency of Agriculture, Fisheries and Environment of

the Union of Comoros. No endangered or protected species were

involved in the survey. Farmers in each zone gave their verbal

consent to be included in the study. Permissions for the blood

sample collection were obtained. The animals were bled without

suffering. Regarding the trade survey, no personal data were

collected, and only information concerning the number of animals

travelling from one island to another was taken into account.

Study zonesThe Comoros islands form an archipelago of volcanic islands

located off the southeastern coast of Africa, east of Mozambique

and northwest of Madagascar. The archipelago is divided between

the sovereign state of the Union of Comoros composed of three

islands named Grande Comore, Moheli, and Anjouan, and the

French overseas department of Mayotte. The tropical climate of

the Comoros islands is characterized by daytime temperatures

around 26uC at sea level, with limited variation during the year,

and by annual heavy rainfall (2,679 mm) with two seasons: a

humid season from November to April, and a dry season from

May to October.

Based on the results of a previous RVFV antibody prevalence

study in 2009 [18], six zones were selected in the Union of

Comoros (Figure 1). Four zones were selected on the island of

Grande Comore: zones 1 and 2 located in the center of the island

where low RVFV antibody prevalence was found, and zones 3 and

4 located in the south with high RVFV antibody prevalence [18].

Zones 2 and 4 are located along the coast (0–200 m above sea

level (asl.)) where ruminants are mostly goats stall reared or

ranging free within and outside villages. Zones 1 and 3 are located

at a moderate altitude (500–650 m asl.) where ruminants are

mostly cattle reared in pastures (zone 1) or raised in stalls in the

forest (zone 3).

Zone 5, which was located on the southern coast of the island of

Moheli, was selected because of its highest RVFV antibody

prevalence observed during the 2009 survey [18]. On this island,

cattle are reared in stalls on an old coconut plantation. Finally, in

zone 6 located close to the airport on Anjouan island, cattle were

raised in stalls in vegetable production areas.

Animals and samplingFive ml of whole blood was collected from the jugular vein of

goats and cattle in Vacutainer tubes (Becton Dickinson, USA).

Samples were allowed to clot at 15uC and serum was separated

from whole blood by centrifugation; samples were stored in liquid

nitrogen in the field and at 280uC in the laboratory.

Livestock longitudinal surveyThe livestock longitudinal survey was conducted in the six

separate zones detailed in Figure 1. From 20 to 30 ruminants were

individually identified in each zone using ear tags. The number of

animals sampled per zone was based on the previous survey, with

a RVFV antibody prevalence ranging from 20% to 50% [18] with

70% relative precision [23]. To avoid colostral immunity, animals

were selected as follows: cattle were between 10 months and one-

year of age, and small ruminants were between three to eight

months of age. Animals were sampled monthly from April 2010 to

Author Summary

Rift Valley fever (RVF) is a viral disease transmitted bymosquitoes to ruminants. The disease may affect humansand has a great impact on the economy of the affectedcountry. RVF occurs mostly in African countries, butepidemics have been reported in Madagascar and in theArabian Peninsula. In the Union of Comoros, RVF virus(RVFV) has been suspected of continuously circulatingsince 2009 without any notifications of the typical clinicalsigns by the Comorian Animal Health Services. From April2010 to August 2011, we conducted a livestock longitu-dinal survey in Grande Comore, Moheli and Anjouan. Ourstudy aimed to detect RVFV-specific antibody acquisitionsin cattle and goats. Simultaneously, a longitudinal ento-mological survey was conducted to describe the diversityof mosquitoes in the study zones and ruminant trade-related information was collected. Our investigationsshowed that Comoros ruminants acquired RVFV-specificantibodies all along the year and particularly in Moheliduring the dry season. Our findings suggest a complexRVFV epidemiological cycle in the Union of Comoros withprobable inter-islands differences in RVFV circulationpatterns. The disease appears to be endemic in Moheliand potentially Anjouan, but the persistence of the diseasein Grande Comore could be correlated with trade in liveanimals with the eastern coast of Africa.

RVFV Circulation in the Union of Comoros


August 2010 and every four months from August 2010 to August

2011. The first series of serological tests determined the RVF

serological status of each sampled animal. Only RVFV antibody

negative animals were included in the livestock longitudinal survey

and continued to be sampled until their IgG RVFV antibody

positive status and were then excluded from the study. When

possible, new ruminants were included in the study to substitute

lost, dead or RVFV IgG positive animals.

Livestock cross-sectional surveyThe RVFV antibody prevalence based on the different study

zones was estimated in August 2011. The sample size was based on

the previously estimated prevalence [18] with a relative precision

of 20% and a confidence level of 95% giving a required minimum

of 385 animals to be collected [23]. Without any particular

Comorian livestock census, animals were selected on the farmer’s

willingness to cooperate during the study.

Longitudinal entomological surveyBlood-sucking insects were sampled every four months from

November 2010 to August 2011 along with the longitudinal

serological survey using double-net goat baited traps placed from

4:00 pm to 10:00 am. The sampling was carried out for three

consecutive days in the study zones numbered 1, 3, 5 and 6

(Figure 1). No sampling was performed in zones 2 and 4 for

logistic reasons.

Environmental dataIn order to generate hypotheses on potential associations

between the estimated RVFV incidence and prevalence with

environmental risk factors for RVF infection, we collated climatic

variables [24]. Two remotely-sensed MODerate-resolution Imag-

ing Spectroradiometer (MODIS) data sets were sourced from the

National Aeronautics and Space Administration (http://modis.

gsfc.nasa.gov/), namely the Daytime Land Surface Temperature

(DLST) and the Nighttime Land Surface Temperature (NLST),

both with spatial and temporal resolution of 1 km and 8 days. In

addition, rainfall data were obtained from the Malaria Early

Warning System (MEWS) program, freely available in the MEWS

repository (http://iridl.ldeo.columbia.edu/expert/SOURCES/.

NOAA/.NCEP/.CPC/.FEWS/.Africa/.TEN-DAY/.RFEv2/.est_

prcp/), with a spatial and temporal resolution of 11 km and 10

days respectively. DLST, NLST and rainfall values were extracted

within a 5-km radius buffer around each farm corresponding to

the maximum daily distance for cattle (grazing and watering). For

each sampled animal that became RVFV antibody positive,

Figure 1. Location of the study zones. Location of the sampling sites (serological survey, entomological trapping and trade analysis). A landingzone is an area on the shore where boats drop passengers and animals off or pick them up.doi:10.1371/journal.pntd.0003045.g001



http://modis.gsfc.nasa.gov/

http://modis.gsfc.nasa.gov/

http://iridl.ldeo.columbia.edu/expert/SOURCES/.NOAA/.NCEP/.CPC/.FEWS/.Africa/.TEN-DAY/.RFEv2/.est_prcp/



MODIS and MEWS data recorded at the time of the serocon-

version in the zone concerned were compared with MODIS and

MEWS data recorded at the same time in the other zones.

Trade surveyThe aim of the trade survey was to estimate the movement of

live animals between continental Africa and the Comoros

archipelago and among the islands of the archipelago themselves.

To date, only approximate figures are known without any

quantitative data available [17]. The number of imported

ruminants was collected monthly between November 2010 and

August 2011 as follows i) the local veterinary authorities provided

records of animal movements through the official ports of Moroni

(Grande Comore), Fomboni (Moheli), and Mutsamudu (Anjouan),

ii) one interviewer per island had the task of identifying undeclared

animal arrivals on the coast, either in the field or from information

provided by village chiefs.

Laboratory testsDetection of RVFV antibodies. Sera were first tested by

two ELISAs (enzymelinked immunosorbent assay) the Immuno-

globulin (Ig) G IDScreen RVF Competition ELISA (IdVet,

France) and the IgM capture ELISA [25]. To confirm their

status, each of the RVFV ELISA antibody positive samples and

some randomly chosen RVFV ELISA negative samples were

tested using the virus neutralizing test (VNT), considered as the

gold standard method described by OIE [26]. Briefly, duplicates

of two-fold serial dilutions of sera starting from 1:5 were added to

100 TCID50 of Smithburn RVFV in 96-well microtiter plates and

incubated for 1 h at 37uC. Next, 100,000 Vero cells were added

to each well and the plates were incubated with 5% CO2 for 5–6

days at 37uC. Titers were expressed as the inverse highest

dilutions giving 50% of CPE. A positive control serum was

included. A serum sample with a titer of 1:10 or higher was

considered seropositive.

Morphological identification of insects. Specimens were

collected by direct aspiration with a home-made vacuum system

and anesthetized with chloroform. Each specimen was morpho-

logically identified by microscopy in the field. Insects were pooled

(1 to 10 individuals), per species per trap and per zone and stored

in liquid nitrogen in the field and at 280uC in the laboratory.

Engorged females were not included in the pool.

Detection of RVFV in insects. The pooled insects were

ground up with 400 ml of PBS 1X (Phosphate Buffered Saline)

twice for 30 seconds with two 3-mm diameter stainless beads using

the TissueLyser system (Loudet, France) and transferred to a 96-

well plate. Total RNA was extracted with the Biomek NX robot

(Beckman Coulter, USA) using the NucleoSpin 96 Virus kit

(Macherey-Nagel, Germany). For RVFV RNA detection, the L-

Segment based SYBR-Green real time PCR was used [27,28].

Ten-fold serial dilutions of a Smithburn strain which contained

108 TCID50/ml were used as the standard curve for plate

validation.

Data analysis and statisticsAll statistics were performed using R.3.0.1 [29]. For both

Fisher’s exact test and the Student-t test, a value of P,0.05 was

considered significant.

A seroconversion was defined as an animal found with either a

positive IgM ELISA result or a positive IgG ELISA result or both

following a previous negative RVFV ELISA sample result.

Incidence rates and instantaneous risk of infection. The

rate of instantaneous risk of infection (Txi) is described as:

Txi~Number of seroconverted animals

Number of animals in an at-risk period

The incidence rate was determined using an Access database in

the Laser format (available at http://livtools.cirad.fr/) [30–32] by

calculating the instantaneous risk of infection (the risk that an

animal will be infected in a given period) [30] taking into account

the risk of seroconversion, death or lost animals. The number of

animals in an at-risk period represents the total number of animals

in a susceptible period of RVFV infection.

Incidence and seroprevalence analysis. To assess whether

location had an effect on the RVFV antibody prevalence, different

zones with specific ecosystems were included in the follow-up

study. A Fisher’s exact test was used to compare the difference in

incidence of RVFV antibody acquisition and RVFV antibody

prevalence between zones and islands. Incidence was analyzed and

hypotheses were proposed about relationships with environmental

and climate conditions.

Analysis of entomological data. A trapping session with no

rain and/or wind was included in the data analysis even if no

blood-sucking insects were collected. Student’s t-Test was used for

statistical analysis.

Trade survey. The movements of animals recorded between

the three islands of the Union of Comoros were mapped, as well as

their potential connection with continental Africa, Mayotte and

Madagascar.

Results

Livestock longitudinal survey in relation withenvironmental data

A total of 191 ruminants (88 cattle and 103 goats) were included

in the livestock longitudinal survey: 135 animals in Grande

Comore, 27 in Moheli and 29 in Anjouan. Detection of RVFV

antibodies (IgM and IgG) was performed by ELISA for a total of

849 serum samples over the duration of study.

Table 1 presents by date and per zone the number of animals

that acquired RVFV antibodies over the duration of the livestock

longitudinal survey. A total of 15 animals out of the 191 sampled

acquired RVFV antibody during the study. Each of the 13

RVFV IgG ELISA positive samples were confirmed by VNT.

Only one RVFV IgM ELISA positive sample was not confirmed

by VNT (July 200, Moheli). This animal was confirmed RVFV

IgG ELISA positive and VNT positive four months later in

November 2010. Out of the 112 RVFV IgG ELISA negative

samples randomly chosen, all were found negative by VNT.

RVFV IgM antibodies acquisition was detected in three animals

and RVFV IgG antibodies acquisition in 12 animals. Only one

RVFV IgM ELISA positive animal in Moheli converted to

RVFV IgG antibodies. The two others RVFV IgM ELISA

positive ruminants were lost or slaughtered before the next

sampling session (Table 1). Nine out of the 15, which acquired

RVFV antibody, were recorded in Moheli, five in Grande

Comore and one in Anjouan. Nine out of those fiftteen occurred

during the dry season (six in Moheli, one in Anjouan, two in

Grande Comore).

The overall annual incidence of RVFV antibody acquisition for

the Union of Comoros was estimated at 17.54% (n[animal risk

time] = 91), with a 95% confidence interval (CI) [8.95–26.14])

(Table 2). A significant difference was found when incidence of

RVFV antibody acquisition was compared between zones (Fisher

exact test, p,0.001) or between islands (Fisher exact test, p,



http://livtools.cirad.fr/

0.001) (Table 2). Zone 5 (Moheli) incidence of RVFV antibody

acquisition (72.3% 95% CI [0.255–1.000]) was significantly higher

than in others zones (Table 2).

The statistical analysis did not reveal any significant difference

in incidence of RVFV antibody acquisition between the rainy

(from November to April,) and the dry season (from May to

October) either for the Union of Comoros as a whole or per zone

(Table 2). DLST, NLST (MODIS data) and cumulative rainfall

(MEWS data) were similar in all six zones at the time of fourteen

out of fifteen seroconversions occurred. There was one exception

when the last RVFV seroconversion was recorded in Grande

Comore in May 2011 (zone 3). Between March and May 2011,

DLST, NLST and cumulative rainfall recorded in Grande

Comore (29uC, 25uC and 730 mm respectively) were higher than

those recorded in Moheli and Anjouan at the same time (DLST :

24uC, NLST : 22uC and cumulative rainfall : 420 mm). No RVFV

seroconversion was recorded on Moheli and Anjouan during this

period.

Livestock cross-sectional studyIn August 2011, to determine the RVFV antibody prevalence, a

total of 275 ruminant samples (i.e. 163 cattle and 112 goats) were

tested for the presence of RVF IgG antibodies. A total of 37

ruminants (20 cattle and 17 goats) came from the longitudinal

follow-up study and 238 ruminants (143 cattle and 95 goats) were

randomly selected in the six separate study zones.

The overall RVFV antibody prevalence in the Union of

Comoros study zones in 2011 was 27.6% (n = 275, 95% CI, [22.3–

32.9]). We found a significant difference of RVFV antibody

prevalence between islands (Fisher exact test, p = 0.007), with a

higher RVFV antibody prevalence in Moheli (45.8%, 95% CI,

[33.7–57.9], Table 3).

Insect trapping and RVFV detectionTwelve trapping days were conducted in each of the four zones

under study (zones 1, 3, 5 and 6, see Figure 1). Blood-sucking

insects were trapped in five out of the twelve trapping days in

central Grande Comore (zone 1), in eight trapping days in

southern Grande Comore (zone 3), eleven trapping days in Moheli

(zone 5), and in seven trapping days in Anjouan (zone 6) (Table 4).

Out of the 1,568 blood sucking insects caught with the double-net

goat baited trap, 1,548 were identified as mosquitoes and 20 were

identified as Stomoxys niger. A total of 1,133 insects were collected

in Moheli (zone 5), 291 in Anjouan (zone 6), 108 in southern

Grande Comore (zone 3) and 36 in central Grande Comore (zone

1). Although the number of comparisons was not large, the

average number of trapped mosquitoes per trapping day per zone

was significantly higher in Moheli (average was 113 insects) and

Anjouan (average was 42 insects) compared to Grande Comore

zones 1 (average was 7 insects) and zone 3 (average was 14 insects)

(Table 5).

The diversity and number of blood-sucking insects caught with

the double net goat baited trap per trapping day per zone are

presented in Table 4. A total of seven genera and 16 species were

caught of which 14 could be morphologically identified. Fifteen

species out of the 16 caught were collected in Moheli (zone 5), nine

species were collected in Anjouan (zone 6), three and eight in

central and southern Grande Comore respectively (zone 1 and

zone 3). Eighty-seven percent of the total number of insects caught

belonged to three species with 52% belonging to two Eretmapo-dites species (E. quinquevittatus and E. subsimplicipes,) and 35% to

Aedes cartroni.No RVFV RNA was detected in any of the 442 pools tested.

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Trade surveyThe study highlighted movements of live ruminants between the

three islands of the Union of Comoros, the African mainland,

Mayotte and Madagascar (Figure 2). Data recorded by veterinar-

ians and technicians showed movements of live ruminants from i)

the east coast of Africa to Union of Comoros and ii) between the

three islands of the Union of Comoros (Figure 2A.). Animals were

observed being landed on beaches without any controls or in

secondary ‘‘ports’’ like Chindini in the south of Grande Comore.

Figure 2B represents the dynamics of live animal importations in

Union of Comoros from May 2010 to July 2011. We recorded up

to ten fold more ruminants imported in Grande Comore than in

Moheli or Anjouan.

Discussion

Rift Valley fever was detected for the first time in Grande

Comore in the human population in 2007 [14] and in livestock in

2009 [18]. Our study demonstrates that RVFV is still circulating

in the Union of Comoros despite of the absence of apparent

clinical signs in livestock.

Fifteen RVFV seroconversions were observed in the Union of

Comoros between 2010 and 2011 giving an overall incidence of

RVFV antibody acquisition of 17.5%. These results suggest

continuous circulation of RVFV on the three islands. However,

significant differences in incidence were observed between islands

(p,0.001). The incidence of RVFV antibody acquisition was

higher in Moheli (72.3%) than in Anjouan (5.8%) and in Grande

Comore (8.2%). This is in accordance with differences in RVFV

antibody prevalence between the Union of Comoros islands

recorded in 2009 and 2011. In 2011, RVFV antibody prevalence

in Anjouan was still below the one in Grande Comore, whereas

RVFV antibody prevalence remained the highest in Moheli.

However, in Grande Comore and Anjouan RVFV antibody

prevalence in 2011 appeared to have decreased whereas in

Moheli, RVFV antibody prevalence remained similar to the level

recorded in 2009, despite herd replacement estimated at 12% (L.

Cavalerie, personal communication). These results suggest the

existence of island specific RVF circulation patterns.

Seasonality of the incidence of RVFV antibody acquisition

needs to be explored. The Comorian livestock farming character-

istics (small herd size and small total number of ruminants) as well

as the field issues did not allow a sufficient number of young

ruminants (nrisk too small) reducing the power of the statistical

analysis.

No clinical signs were reported in the Union of Comoros during

the period of our study, as reported in Madagascar, Tanzania, and

Mozambique in recent years [33–35], but the fifteen seroconver-

sions observed suggest that RVFV could be circulating in the

Comorian environment thanks to local mosquito-mammalian host

cycles even if the numbers of caught mosquitoes were not large nor

positive for RVF RNA. Out of the 1,568 blood-sucking insects

caught, none were found to be RVFV RNA positive by PCR but

in the absence of RVF outbreaks, chances of detecting RVFV in

vector populations are known to be very low [36]. In 1978,

Bruhnes described 30 mosquito species in the Union of Comoros

[22]. Four of them: Ae. aegypti, Ae. fowleri, Ae. circumluteolus and

Cx. quinquefasciatus are considered as RVFV potential vectors

because the virus has been already isolated in these species in the

field and because of their capacity to transmit RVFV under

laboratory conditions [37–39]. All these species, except Ae.fowleri, have been caught at least on one island during our study,

suggesting a role for this mosquito species to be involved in the

transmission cycle on each of the islands. Five other mosquito

species caught during our study, Er. quinquevittatus, An.arabiensis, M. uniformis, An. coustani and Ae. simpsoni were

previously identified as RVFV RNA positive by PCR in the field

[40–44]. An. coustani and Ae. simpsoni were found RVFV RNA

carrier for the first time in the Indian Ocean area: respectively in

Madagascar in 2011 and in Mayotte in 2009 [43,44]. Thus, some

of these mosquito species may play a role in RVFV transmission in

the Union of Comoros. Geological inaccessibility, sampling design

and climatic conditions likely explain the small number of

specimens caught and the heterogeneity of entomological findings

between islands [45]. These volcanic islands are characterized by a

tropical climate with only slight variations in daily temperatures

and abundant rainfalls, which theoretically should enable popu-

lations of Culicidae species to persist throughout the year.

Table 3. RVFV antibody prevalence in the Union of Comoros, 2011.

RVFV antibody prevalence Statistical analysis [p-value]

Grande Comore Moheli Anjouan

Grande Comore prevalence 0.247

n 174

95% CI [0.183–0.311]

Moheli prevalence 0.458 0.008*

n 48

95% CI [0.317–0.599]

Anjouan prevalence 0.207 0.683 0.013*

n 53

95% CI [0.098–0.317]

Union of Comoros prevalence 0.276 0.007**

n 275

95% CI [0.223–0.329]

CI: stands for Confidence Interval, *Fisher exact test, 2 by 2 comparison, p-value significant if p,0.05, ** Fisher exact test, multiple comparison, p-value significant if p,

0.05.doi:10.1371/journal.pntd.0003045.t003



Nevertheless, each island has its own environmental characteris-

tics, as the age of the three islands decreases westward: Moheli is

2.7360.20 million years old, Anjouan, 1.1860.03 million years

old, and Grande Comore is 0.1360.02 million years old [46].

On Moheli and Anjouan, the oldest islands, the landscape

includes permanent rivers [47] and, as a result, many artificial and

natural breeding mosquito sites exist. Moheli has a wide variety of

natural and artificial sites in which mosquitoes can breed all year

round [47,48]. The presence of clay, resulting from the decompo-

sition of volcanic soils, ensures the presence of abundant surface

water impoundments. It allows the cultivation of irrigated rice hence

and favors the development of diversified mosquito populations

[47]. A greater number of mosquito species were caught in Moheli

(15 species) than in the other islands which is in agreement with

Brunhes’ inventory in 1978, including two mosquito species known

as RVFV potential vectors. Thus, favorable conditions for RVFV

persistence being present a better chance for a possible RVFV cycle

involving vectors and animals is suggested.

The abundance of mosquitoes trapped in Anjouan (zone 6) was

similar to that in Moheli (zone 5) and three mosquito species

known as RVFV potential vector have been caught during our

study. However, RVFV antibody prevalence in Anjouan was the

lowest and appeared to be decreasing. Moreover in 2011, only one

ruminant exhibited a RVFV seroconversion. Anjouan shares some

similar environmental characteristics with Moheli that could allow

mosquitoes to survive all year round but Anjouan has some

characteristics that could limit the circulation of RVFV. For

example, the landscape is comprised of hill slopes and irrigated

field rice is not cultivated on the island. Ruminants are mainly

reared in stalls in the highlands in the eastern part of the island.

For that reason, the probability of contact between infected vectors

and ruminants may be lower in Anjouan than in Moheli and the

maintenance of a vector-ruminant cycle may be harder to get.

More investigations in other cattle-rearing areas are thus needed

to conclude on RVF circulation in Anjouan.

Incidence of RVFV antibody acquisition and the RVFV

antibody prevalence in Grande Comore are hard to explain

based only on entomological parameters. Presence of steep slopes

with decomposed and highly permeable soils characterize Grande

Comore, the youngest island of the country [47]. Surface water is

Table 4. Diversity and number of blood-sucking insects caught with a double baited net per trapping day and per zone, Union ofComoros, 2011.

Grande Comore Moheli Anjouan

Genus species Zone 1 Zone 3 Zone 5 Zone 6 Total number

Stomoxis niger 0 0 11 9 20

Aedes aegypti 1 4 8 15 28

Aedes cartroni 0 0 504 48 552

Aedes circumluteolus 0 0 0 10 10

Aedes simpsoni 0 20 1 0 21

Aedes vittatus 0 3 13 6 22

Anopheles arabiensis 0 0 2 1 3

Anopheles coustani 0 0 4 0 4

Anopheles sp 0 0 25 0 25

Culex carleti 0 1 9 0 10

Culex quinquefasciatus 0 1 12 34 47

Culex sp 0 0 12 0 12

Eretmapodites subsimplicipes/quinquevittatus 35 77 524 168 804

Uranotaenia pandani 0 2 6 0 8

Mansonia uniformis 0 0 2 0 2

Total number of blood-sucking insects caught (effective trapping days*) 36(5) 108(8) 1133(11) 291(7) 1568(31)

*number of effective trapping days, i.e. days with the right climatic conditions (no wind or rain) to catch insects.doi:10.1371/journal.pntd.0003045.t004

Table 5. Comparison of average number of mosquitoes caught per trapping day per zone (Student’s t-Test), Union of Comoros,2011.

Grande Comore Moheli

Zone 1 Zone 3 Zone 5

Anjouan Zone 6 p = 0.005* p = 0.012* p = 0.116

Moheli Zone 5 p = 0.020* p = 0.031* -

Grande Comore Zone 3 p = 0.280 - -

* p-value significant if p,0.05.doi:10.1371/journal.pntd.0003045.t005



Figure 2. Trade in live animals between the Comoros archipelago, Madagascar and East Africa between 2007 and 2012. Trade in liveanimals between the Comoros archipelago, Madagascar and East Africa between 2007 and 2012 (Figure 2a) and imported animal dynamics (Figure2b). Data used to create this map were gathered in surveys, or came from official sources or from personal communications.doi:10.1371/journal.pntd.0003045.g002



rare and only artificial containers (such as tanks and troughs) and

some natural breeding sites (such as coconut shells and hollow

trees) enable the development of Culicidae. Our results were in

accordance with these observations as fewer blood-sucking insects

were caught in Grande Comore when compared to Moheli and

Anjouan. Thus, mosquito abundance in Grande Comore was

likely correlated with the number of breeding sites that appeared

after rainy episodes, as observed for the seroconversions we

detected in Grande Comore following on from a major increase in

cumulative rainfall. Two out of eight mosquito species caught

during our study have been described as RVFV potential vectors.

Consequently, environmental conditions for a local mosquito-

mammalian host cycle could be met after important rainy episodes

but a continuous circulation of RVFV in Grande Comore all year

round is less likely to happen. However, regular introductions of

the virus through the arrival of live animals from Tanzania [49],

Anjouan, and Moheli may play a role in the persistence of RVFV

in Grande Comore.

Analysis of trade in live animals confirmed observations

reported by Cetre et al., in 2012 in an overview of the movement

of live ruminants between east Africa and the Comoros

archipelago, as well as within the archipelago. Per year, more

than 3000 live ruminants are imported from Tanzania (Chief

Veterinary Officer of Comorian Vet services, personal communi-

cation), where RVF is endemic [34]. These animals enter the

Union, mostly Grande Comore, without any quarantine or clinical

examination. The risk of the introduction of new exotic strains of

RVFV is consequently quite high and could affect the country in

the same way as many other diseases in the past [19]. Tanzanian

ruminants are imported for ‘‘great weddings’’ which are usually

celebrated in July and August in Grande Comore. During these

traditional weddings, villagers sacrifice ruminants without any

particular sanitary rules. However, no major cases in humans and

no ruminant seroconversions were reported during the ‘‘great

weddings’’ period during our study but to date, human and

veterinary health surveillance networks remain not very efficient.

Occasional imports of Tanzanian ruminants into Moheli and

Anjouan have also been reported; so new RVFV strains could

have been also introduced on these islands. The regular

introduction of live ruminants from Anjouan and Moheli could

also contribute to the regular introduction of RVFV in Grande

Comore as well.

Rift Valley fever epidemiology in the Union of Comoros is

complex and further virological investigations should help to

explain the origin of the RVFV strain(s) circulating within the

islands. However, based on the results of the present study, RVFV

seems hardly to persist on Grande Comore through a local vector

cycle only but repeated reintroduction of viruses is possible. The

situation regarding Rift Valley fever in Anjouan and Moheli

appeared to look like that in Mayotte, Madagascar, Tanzania, and

Mozambique [33–35,50] where RVFV seroconversions have also

been observed in the dry season without any apparent clinical

signs. These findings could identify Moheli and Anjouan as

endemic areas for RVFV. Given the incidence of RVFV

seroconversions and antibody prevalence, RVFV is more likely

to be circulating in Moheli than in Anjouan. However, additional

data are needed to firmly conclude on the circulation of RVFV in

the Union of Comoros. Wildlife such as bats and lemur species in

our zone should be investigated even though no wildlife reservoir

has been identified in any other country so far [51,52].

Rift Valley fever is still a burden for the Union of Comoros as

new human cases were diagnosed as RVFV positive in 2011 and

in 2012 either by IgM or RVFV RNA detection with clinical signs

[53,54]. The real impact of the disease on human health and on

the national economy is still unknown. Human and veterinary

health networks need to be strengthened including the establish-

ment of quarantine for imported ruminants.

Acknowledgments

This study was conducted in the framework of AnimalRisk-OI, a research

program on emerging animal diseases in the Indian Ocean. The contents

of this publication are the sole responsibility of the authors and do not

necessarily reflect the views of the European Commission. We thank all the

Comoros farmers, veterinarians and veterinary technicians for their

participation in the fieldwork as well as S. Girard. We thank R. Lancelot

for his help with the Laser database and incidence calculation. We thank

W. Wint, D. Rogers and D. Benz of the Spatial Ecology and Epidemiology

Group (University of Oxford) for providing MODIS data. We also thank L.

Cavalerie for her personal remarks on RVF circulation in the Union of

Comoros, C. Sutter for providing the striking image and T. Balenghien for

his critical review.

Author Contributions

Conceived and designed the experiments: MR SL EC. Performed the

experiments: MR SL MS AF CF AS AMK. Analyzed the data: MB SL

MR CCS EC. Contributed reagents/materials/analysis tools: MMO MM

CCS MB SL. Wrote the paper: MR MB SL. Minor revisions of

manuscript: MR SL MB CCS.

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